4-MU was purchased from Nacalai Tesque (Kyoto, Japan) and diluted in dimethylsulfoxide (Wako Pure Chemical Industries, Ltd., Osaka, Japan); the working concentration was 500 µM. The reason for using 500 µM 4-MU was that clear effects of 4-MU could be observed and the cytotoxic effect was low for normal fibroblast cells at a 500 µM concentration (22). Monoclonal phycoerythin (PE)-conjugated anti-human cluster of differentiation (CD)126 antibody (cat. no. 352804); mouse monoclonal PE-IgG1, κ isotype control (cat. no. 400114); monoclonal allophycocyanin (APC)-conjugated anti-human CD130 (gp130) antibody (cat. no. 362005); mouse monoclonal APC-IgG2a, κ isotype control (cat. no. 400221); fluorescein isothiocyanate (FITC)-annexin V (cat. no. 640905); and propidium iodide (PI) (cat. no. 421301) were from BioLegend (San Diego, CA, USA). PE-conjugated polyclonal anti-human type 1 IL-1 receptor (IL-1R) antibody (cat. no. FAB269P) and PE-conjugated goat IgG (cat. no. IC108P) were R&D Systems, Inc., (Minneapolis, MN, USA).

HT1080 human fibrosarcoma cells from American Type Culture Collection (Manassas, VA, USA) were cultured in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Japan Bio Serum, Fukuyama, Japan) and 1% penicillin/streptomycin (Life Technologies) at 37°C in a humidified atmosphere of 5% CO₂.

The clonogenic potency of HT1080 cells was estimated with the colony formation assay. Appropriate numbers of cells were seeded and incubated for 2 h, then subjected to X-ray irradiation at 1–6 Gy in the presence of 500 µM 4-MU followed by incubation for 24 h. After 7–10 days of culture with regular changes of medium cells were fixed with methanol (Wako Pure Chemical Industries, Ltd.) stained by Giemsa (Wako Pure Chemical Industries, Ltd.), and quantified.

Ionizing radiation (IR) was delivered using an X-ray generator (MBR-1520R-3; Hitachi Medical, Co., Tokyo, Japan) with 0.5-mm aluminum and 0.3-mm copper filters at a distance of 45 cm between the focus and target (150 kV, 20 mA, 1.0 Gy/min). During X-ray exposure, the total dose and dose rate were monitored with a thimble ionization chamber placed next to the sample.

To evaluate the expression of the IL-6 receptors CD126 and CD130, and the IL-1α/β receptor type I IL-1R, cells were resuspended in 100 µl Dulbecco's phosphate-buffered saline without CaCl₂ and MgCl (Takara Bio, Inc., Otsu, Japan) containing 5% FBS and PE-conjugated anti-human CD126 antibody (3 µl/106 cells), APC-conjugated anti-human CD130 antibody (3 µl/106 cells), and PE-conjugated anti-human type 1 IL-1R antibody for 15 min at 4°C in the dark. To detect cell death, cells labeled with FITC-annexin V (3 µl/106 cells) were resuspended in annexin V binding buffer (cat. no. 422201; BioLegend) and incubated for 15 min at room temperature in dark with PI (6 µl/106 cells), followed by flow cytometry analysis on FACS Aria instrument (BD Biosciences, Tokyo, Japan).

Total RNA was extracted from HT1080 cells 24 h after irradiation and/or 4-MU administration using the RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), and RNA quality was confirmed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Cyanine (Cy)3-labeled cRNA samples were synthesized from total RNA and hybridized to 8×60K format SurePrint G3 Human GE v2 microarray slides (eArray Design ID=039494) according to the manufacturer's instructions (Agilent Technologies, Inc.). Cy3 fluorescence was detected with a DNA microarray scanner (G2600A SureScan) and processed using Feature Extraction software (both from Agilent Technologies, Inc.).

cDNA was synthesized using a RT kits (Applied Biosystems; Thermo Fisher Scientific, Inc.) and used as a template for PCR in a 20-µl reaction mixture containing 2X SYBR Premix Ex Taq (Takara Bio, Inc.) and 0.5 µM forward and reverse primers (Table I). The reaction was carried out on a real-time PCR system (StepOne Plus; Life Technologies) under the following conditions: 30 sec at 95°C, followed by 40 cycles of 95°C for 5 sec, and 54°C for 30 sec. Target gene expression levels were calculated relative to that of glyceraldehyde 3-phosphate dehydrogenase mRNA (internal control) with the comparative ΔΔCq method (23).

The levels of IL-1α, −1β, and −6 secreted by cells were measured using the Human IL-1α, IL-1β, and IL-6 DuoSet ELISA kits (all from R&D Systems), respectively, according to the manufacturer's protocols. Cells were treated with 500 µM 4-MU and irradiated with 2 Gy X ray, followed by incubation in serum-free medium. Culture medium conditioned for 24 h was used for the ELISA assay. Cytokine concentration was determined per million cells.

Statistical analysis of microarray data was performed using GeneSpring (Agilent Technologies, Inc.). Up- and downregulated mRNAs transcripts were selected based on fold change (>2-fold) of irradiated and/or 4-MU-administered samples relative to control samples. Ingenuity Pathway Analysis (IPA; Qiagen Silicon Valley, Redwood City, CA, USA) was used for functional analysis of each transcript. The significance of differences between control and experimental cultures was evaluated with one-way analysis of variance and the Tukey-Kramer test. Statistical analyses were performed using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA) with the add-on software Statcel v3 (OMS Publishing, Saitama, Japan). P<0.05 was considered to indicate a statistically significant difference.

To investigate whether the anti-inflammatory effect of 4-MU enhances radiosensitization, we evaluated the clonogenic potency of HT1080 cells exposed to 4-MU and/or X-ray radiation with the colony formation assay. The survival of cells treated with 4-MU combined with X-ray irradiation was decreased in a radiation dose-dependent manner compared to X-ray irradiation alone (Fig. 1A). This was confirmed by annexin V and PI staining. Representative dot plots and quantified data are shown in Fig. 1B and C. The number of cells positive for both annexin V and PI was increased 6 h after X-ray irradiation (9.12±0.52%) (Fig. 1C). A comparable increase was observed upon treatment with 4-MU alone for 6 h (10.13±0.03%). On the other hand, there were more annexin V and PI positive cells in the group exposed to both 4-MU and X-ray radiation as compared to either treatment alone (12.58±0.23%). These results suggest that 4-MU enhances the sensitivity of HT1080 cells to the lethal effects of X-ray radiation.

We performed a microarray analysis to determine the signaling pathways affected by 4-MU treatment combined with X-ray irradiation. A total of 2873, 4085, and 3179 genes were differentially expressed in cells treated with 4-MU alone, X-ray alone, and 4-MU + X-ray, respectively. The IPA z-scores revealed that signaling pathways associated with inflammation including IL-8 and −6 and Toll-like receptor signaling were altered by treatment with 4-MU or/and 2 Gy X-ray radiation (Fig. 2A). Moreover, IL-1α and −1β were inactivated in cells treated with 4-MU only (Fig. 2B).

IL-1α, IL-1β, and IL-6 mRNA levels in cells and IL-1α, IL-1β, and IL-6 concentrations in the cell culture supernatant were analyzed by RT-qPCR and ELISA, respectively. IL-1α and −1β transcript levels in cells exposed to X-ray radiation alone were increased about 2-fold relative to the control, whereas the levels in cells treated with 4-MU showed the opposite trend (Fig. 3A). However, IL-1α concentration in the supernatant was 3-fold higher in 4-MU treated as compared to control cultures (1.73±0.21 vs. 5.09±0.48 pg/ml), whereas IL-1α concentration (per 106 cells) in the culture supernatant of cells exposed to both 4-MU and X-ray radiation was higher than that in the radiation-only group (5.71±1.73 vs. 2.20±0.31) (Fig. 3B). On the other hand, the IL-1β concentration in the culture supernatant was correlated with the mRNA expression. X-ray irradiation increased IL-6 mRNA level 1.7-fold relative to the control (Fig. 3A); however, the level was decreased 0.8-fold by treatment with 4-MU combined with X-ray irradiation. IL-6 level in the culture supernatant of cells exposed to radiation alone was 2-fold higher than that in the control group (2.84±0.21 vs. 5.95±0.11 pg/ml) (Fig. 3B), whereas concentrations for cells treated with 4-MU with or without X-ray irradiation were similar to that of control cultures (2.05±0.36 and 2.39±0.21 pg/ml, respectively). This suggests that the increase in IL-6 expression and release induced by X-ray irradiation was reversed by 4-MU treatment which suppressed inflammation by inhibiting IL-1β production.

To investigate whether IL-1α, −1β, and −6 receptors are suppressed by 4-MU, we examined the expression of the cognate surface receptors [type 1 IL-1R (CD121a), CD126, and CD130, respectively] by flow cytometry. The mean fluorescence intensity of CD121a on cells exposed to X-ray radiation alone was higher than that of control cells (59.88±18.3 vs. 74.29±10.3, respectively) (Fig. 4A and D). On the other hand, the mean fluorescence intensity of CD121a in the 4-MU only (55.22±4.38) and 4-MU/X-ray radiation combination (62.79±3.72) groups did not differ significantly from that of the control group (P=0.429, 0.268, respectively), suggesting that the increase in CD121a expression induced by X-ray irradiation was marginally suppressed.

The mean fluorescence intensity of CD126, the ligand binding protein for IL-6, was unaltered by X-ray irradiation relative to the control (74.48±5.74 vs. 96.17±14.1). 4-MU treatment reduced the signal intensity to about half that of the control group (4-MU alone: 56.54±6.12; 4-MU combined with X-ray: 52.11±3.71) (Fig. 4B and D). On the other hand, the mean fluorescence intensity of CD130, the signal transducer for IL-6 receptor, was downregulated by 2 Gy X-ray irradiation as compared to the control (260.40±11.4 vs. 295.76±1.46) and was further decreased by 4-MU administration (4-MU alone: 239.42±9.99; 4-MU combined: 249.57±13.5) (Fig. 4C and D). These results indicate that expression of IL-6 receptors were suppressed by 4-MU treatment.